Membrane Perforation Thermodynamics of a Pore-Forming Toxin Explored by Molecular Dynamics Simulations

Abstract

Pore-forming toxins constitute one of the most spectacular attack weapons of bacteria. Secreted in soluble form, they undergo large conformational changes after different stimuli, allowing them to create a pore in the host's membrane. For instance, upon endocytosis, the pentameric TcA proteins experience a large conformational change due to the acidification of the endosome. The contraction of a 60-residues-long linker peptide is proposed to drive the syringe-like injection of a helical domain into the membrane, which subsequently opens to create a pore. Unfortunately, the thermodynamics behind this process are poorly understood. Here, we used molecular dynamics and free energy calculations to characterize the thermodynamics associated with the membrane perforation of TcdA1 toxin from Photorhabdus luminescens. Our all-atom free energy calculations showed that the contraction of each linker peptide in the pentamer is associated with a gain of ∼10 kcal/mol. Interestingly, this change is largely independent from pH, suggesting that the acidification of the endosome is only required to initiate the conformational change. In addition, we performed coarse-grained free energy calculations of the syringe-like perforation of a POPC model membrane. The results show that the process is favorable by ∼8 kcal/mol. However, a barrier of ∼35 kcal/mol, originated by the opening of the outer leaflet of the membrane, acts against membrane perforation. Combined, this suggests that the effect of the linker contraction is mainly kinetic, decreasing the effective barrier for the perforation process. Interestingly, our all-atoms simulations of the membrane-embedded protein show that several lipidic head groups from the lower leaflet intercalate between the helices of the protein, stabilizing the open conformation. Taken together, this suggest that the pore opening is induced by the interaction of the protein with the membrane, and occurs only upon full perforation.